Fragment Bb of bovine complement factor B: stimulatory effect on the microbicidal activity of bovine monocytes.

We have previously shown that the Bb fragment of bovine complement factor B activates bovine monocytes and neutrophils. The activation was demonstrated by the enhanced uptake of 3H-deoxyglucose. To investigate the potential effect of fragment Bb on the microbicidal activity of bovine monocytes, a direct method was used. This method involves an initial ingestion period at 37 degrees C followed by repeated washing. The decrease in the total number of viable intracellular Staphylococcus aureus during the reincubation of the bacteria with bovine monocytes determines the intracellular killing. Maximal intracellular killing was seen when the monocytes containing the ingested S. aureus was incubated with fresh bovine serum (mean +/- SEM = 73.4 +/- 1.4%). On incubation of the monocytes, containing the ingested bacteria with heat-inactivated bovine serum, 32.5 +/- 0.7% of the intracellular bacteria were killed. When affinity-purified bovine factor Bb was added to the heat-inactivated serum, the intracellular killing capacity was almost restored (65.8 +/- 1.5%). When monocytes were incubated with medium alone, they killed 22.4% of the intracellular microorganisms. When fragment Bb (25 micrograms/mL) was added to the medium, the intracellular killing of S. aureus doubled (46 +/- 1.29%). We conclude that the Bb fragment of bovine complement factor B stimulates bovine monocytes in their microbicidal activity.     

Electron microscopic investigation of intracellular events after ingestion of Rhodococcus equi by foal alveolar macrophages

It has been suggested that R. equi causes pulmonary disease in foals by persisting within the lung as a facultative intracellular parasite of alveolar macrophages. This paper describes an ultrastructural study of the intracellular events after ingestion of R. equi by foal alveolar macrophages, in an attempt to determine the mechanism of intracellular survival of R. equi. Secondary lysosomes of alveolar macrophages recovered from foals by bronchoalveolar lavage were labelled with electron-dense ferritin, and the cells were challenged with either viable or formalin-killed R. equi. After 0-, 3-, 8- or 24-h incubation, the cells were fixed and processed for electron microscopy. There was no evidence of phagosome-lysosome fusion after ingestion of either viable or non-viable R. equi by foal alveolar macrophages. Rhodococcus equi persisted and multiplied within dilated phagosomes, which were often lined by elongate microvillous structures. After 24-h incubation, 75% of the ingested bacteria were still structurally intact. Macrophages with ingested viable R. equi were irreversibly damaged and released intracellular bacteria into the surrounding medium. These data confirm that R. equi is a facultative intracellular parasite of foal alveolar macrophages and is able to persist and multiply within the phagosome, apparently inhibiting phagosome-lysosome fusion by some as yet unknown mechanism.     

Extracellular signal-regulated kinase (ERK) activation in chicken heterophils stimulated with phorbol 12-myristate 13-acetate (PMA), Formyl-methionylleucyl-phenylalanine (fMLP) and lipopolysaccharide (LPS)

The signaling pathways leading to the activation of extracellular signal-regulated kinase (ERK) by phorbol 12-myristate 13-acetate (PMA), formyl-methionylleucyl-phenylalanine (fMLP) and lipopolysaccharide (LPS) in chicken heterophils were examined. To determine the mechanism of ERK's activation and its relation with the influx of calcium ions, heterophils were stimulated by PMA, fMLP and LPS. ERK was not activated by fMLP. LPS- and PMA-stimulated activation of ERK, based on Western blotting with antibodies against the phosphorylated form of ERK, was attenuated by the pretreatment of cells with the intracellular calcium chelator BAPTA/AM (1,2-bis (o-aminophenoxy) ethane-N,N,N',N'-tetraacetic acid) but not with the extracellular calcium chelator EGTA (glycol-bis(2-aminoethylether)-N,N,N',N'-tetraacetic acid). Exposure of cells to the protein kinase C (PKC) inhibitor GF109203X inhibited the LPS- and PMA-stimulated phosphorylation of ERK in a concentration-dependent manner. The LPS-stimulated phosphorylation was inhibited by pretreatment with the phospholipase C (PLC) inhibitor U73122 but not the phosphatidylinositol 3-kinase (PI₃K) inhibitor LY294002. These results indicate that the LPS-induced phosphorylation of ERK in the chicken heterophils is mediated by PLC, PKC and intracellular calcium, and the PMA-stimulated phosphorylation is dependent on intracellular calcium ion and PKC.     

Propionate induces pH(i) changes through calcium flux, ERK1/2, p38, and PKC in bovine neutrophils

Propionate is a short-chain fatty acid produced under normal physiological conditions in the rumen of cattle. It is also involved in the inflammatory process and neutrophil function via calcium release, reactive oxygen species and intracellular pH (pH_(i)) changes. This study examined the effect of propionate on the pH_(i) of bovine neutrophils; specifically if pH_(i) changes are controlled by calcium flux, and the mitogen-activated protein kinase (MAPK) pathway. Propionate caused rapid intracellular acidification and sustained alkalinization in bovine neutrophils loaded with 2′,7′-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein acetoxymethyl ester (BCECF-AM), a fluorescent indicator of pH_(i). The acidification phase seems to be controlled by intracellular calcium release and p38 MAPK pathway. The pH recovery phenomenon was mediated by an amiloride-sensitive Na⁺/H⁺ exchanger and H⁺ channel, and was inhibited by UO126 (an ERK1/2 MAPK phosphorylation inhibitor), Gö6850 (a PKC inhibitor) and calcium chelating. Ionomycin, a calcium ionophore, induced intracellular acidification and sustained alkalinization. The intracellular acidification was strongly inhibited by BAPTA-AM (an intracellular calcium chelator) and SB203580 (a p38 MAPK inhibitor). In addition, the intracellular alkalinization was reduced by EGTA (a calcium chelator), UO126, LY294002 (a PI3K inhibitor) and Gö6850. Propionate did not increase superoxide production, however it reduced the superoxide production induced by platelet-activating factor (PAF), and increased the release of superoxide induced by ionomycin. Our results suggest that propionate-induced intracellular acidification is mediated by intracellular calcium release and p38 MAPK activation, and that pH recovery is controlled via ERK1/2 MAPK, PKC and calcium entry in bovine neutrophils.     

Nitric oxide stimulates IP3 production via a cGMP/ PKG-dependent pathway in rat pancreatic acinar cells

In an attempt to explore the functioning of nitric oxide (NO) in pancreatic exocrine cells, we have recently obtained several lines of circumstantial evidence indicating that one of molecular targets of NO is phospholipase C (PLC), the activation of which leads to an increase in the cytosolic Ca2+ concentration ([Ca2+]i) via inositol 1, 4, 5-trisphosphate, IP3. However, whether IP3 is actually produced by NO has not yet been substantiated. The present study was therefore designed to directly measure the intracellular IP3, concentration ([IP3]i) for better understanding of the underlying mechanisms with the help of pharmacological tools. [IP3]i was measured using a fluorescence polarization technique (HitHunter). We obtained the following results: 1) varying concentrations of an NO donor, sodium nitroprusside (SNP), elevated [IP3]i, 2) this elevation was completely inhibited in the presence of the soluble guanylyl cyclase (sGC) inhibitor, 1H-[1, 2, 4] oxadiazolo [4, 3-a] quinoxalin-1-one (ODQ), 3) varying concentrations of the cGMP analogue, 8-Br-cGMP, also increased [IP3]i, 4) the cGMP analogue-induced IP3 production was abolished by pretreatment with either a PLC inhibitor, U73122, or a G-protein inhibitor, GP2A, and 5) KT5823, a potent and highly selective inhibitor of cGMP-dependent protein kinase G (PKG), also abolished the IP3 production induced by 8-Br-cGMP. These results suggest that the NO-induced [Ca2+]i increase is triggered by an increase in [IP3]i located downstream from intracellular cGMP elevation. In this intracellular pathway, each sGC, cGMP-dependent PKG, G-protein and PLC were suggested to be involved. The present work provides new insights into the intracellular signaling accelerated by NO. NO triggers a [Ca2+]I increase via cGMP and IP3 in pancreatic acinar cells.     

Relationship between the chemiluminescent response of bovine neutrophils and changes in intracellular free calcium concentration.

The relationship between luminol dependent chemiluminescent (LDCL) response and changes in intracellular free Ca2+ concentrations in the bovine neutrophils was evaluated. LDCL responses and changes in intracellular Ca2+ concentrations of neutrophils were clearly detected by the stimulation with opsonized zymosan (OPZ), concanavalin A(ConA), heat-aggregated IgG (H-agg.IgG) and phorbol myristate acetate (PMA). Patterns of LDCL responses and intracellular Ca2+ of neutrophils showed characteristic features for each stimulant. PMA was a weak stimulant of the intracellular Ca2+ concentration, whereas it was a strong stimulant of LDCL response. Con A strongly stimulated an increase in the intracellular Ca2+ concentration, but was a weak stimulant of LDCL response. LDCL response of intracellular Ca(2+)-depleted neutrophils treated with ionomycin, stimulated with each stimulant was inhibited markedly without extracellular Ca2+. The sustained phase of intracellular Ca2+ concentrations stimulated with OPZ was inhibited significantly (P < 0.05) by the preincubation with anti-CD18 antibody, whereas the transient phase of intracellular Ca2+ concentrations was not inhibited. These results indicate that LDCL response is regulated at least in part by the elevation of the intracellular Ca2+, and a rise in intracellular Ca2+ concentration, which may be mediated by specific receptors appears to be essential in the LDCL response of bovine neutrophils.     

Determination of intracellular cytokines IFN-γ and IL-4 in canine T lymphocytes by flow cytometry following whole-blood culture

This report describes a whole-blood flow cytometric method for the determination of intracellular cytokines IFN-γ and IL-4 in canine T lymphocyte subpopulations. Conjugated anti-cytokine antibodies and commercially available reagents for cell fixation and permeabilization were used. Canine peripheral blood was cultured with a combination of phorbol-12-myristate-13-acetate (PMA) and ionomycin to promote cytokine synthesis in each cell, along with monensin to increase the sensitivity of the method by retaining IFN-γ and IL-4 within the cell to detectable levels. The optimum concentrations of PMA and ionomycin were determined. Maximum IFN-γ expression from both CD4+ and CD8+ T lymphocytes was detected after 6 h of incubation of cell culture, while maximum IL-4 production took 6 h from CD4+ cells and 4 h from CD8+ cells. This method is a simple immunologic technique for measuring intracellular cytokines which could be of value in the investigation of canine immunological response mainly in various intracellular and extracellular infections, since IFN-γ and IL-4 are considered key cytokines activating the cellular and humoral immunity, respectively.     

Mitogen and antigen-specific induction of lymphoblast transformation in cats with subclinical toxoplasmosis.

Lymphoblast transformation in whole blood was assessed by 3H-thymidine incorporation after stimulation by concanavalin A and Toxoplasma gondii secretory and intracellular antigens in samples from cats with experimentally induced toxoplasmosis. Toxoplasma gondii-specific immunoglobulin M, immunoglobulin G, and circulating antigens were also measured throughout the study period. Lymphocytes from all cats were responsive to concanavalin A pre- and post-inoculation with T. gondii. Suppression of mitogen-stimulated lymphoblast transformation during the course of infection was not observed. Both the secretory and intracellular antigens stimulated lymphoblast transformation in many cats from Week 2 to Week 52 post-inoculation. Lymphoblast transformation was stimulated more frequently by intracellular antigens (66.25%) than by secretory antigens (48.75%). Lymphoblast transformation was not induced by T. gondii antigens in any cat prior to appearance of T. gondii-specific antibodies in serum or during the oocyst shedding period. Cats with persistent antigenemia had the most consistent lymphoblast transformation results induced by T. gondii-specific antigens.     

The evaluation of the potential of botulinum C3 enzyme as an exogenous differentiation inducing factor to neurons

Botulinum C3 enzyme produced by Clostridium botulinum type C and D strains modifies Rho proteins. In a previous study, we observed that the LDH isozyme pattern of neurons treated with C3 enzyme was different from that induced with endogenous growth factor of neurons such as NGF [21]. This type of change is considered to have an advantage in the medical use of C3 enzyme for neural disorder. To determine the functional similarity of C3-treated neurons to control and NGF-treated neurons, we examined the responses of C3-treated neurons to various drugs, including some neurotransmitters, by measuring the rise of intracellular Ca ions into the neurons. The time course of the rise of intracellular Ca ions induced by high concentration of potassium in the C3-treated neurons was similar to that in the NGF-treated neurons. The C3-treated neurons responded to glutamic acid, aspartic acid, kainic acid, gamma-aminobutylic acid, muscarine and ACh with similar time courses and magnitudes as the control neurons. These results suggest that the C3 enzyme induces the functional differentiation of neurons, and that C3 enzyme has the potential for the medical use as an exogenous differentiation-inducing factor of neurons.     

A Trial to Cryopreserve Immature Medaka (Oryzias latipes) Oocytes after Enhancing Their Permeability by Exogenous Expression of Aquaporin 3

Fish oocytes have not been cryopreserved successfully, probably because it is difficult to prevent intracellular ice from forming. Previously, we have shown in medaka that immature oocytes are more suitable for cryopreservation than mature oocytes or embryos, in terms of permeability. We have also shown in immature medaka oocytes that the exogenous expression of aquaporin 3 (AQP3), a water/cryoprotectant channel, promotes the movement of water and cryoprotectants through the plasma membrane. In the present study, we attempted to cryopreserve immature medaka oocytes expressing AQP3. We first examined effects of hypertonic stress and the chemical toxicity of cryoprotectants on the survival of the AQP3-expressing oocytes. Exposure to hypertonic solutions containing sucrose decreased the survival of oocytes, but the expression of AQP3 did not affect sensitivity to hypertonic stress. Also, AQP3 expression did not markedly increase sensitivity to the toxicity of cryoprotectants. Of the four cryoprotectants tested, propylene glycol was the least toxic. Using a propylene glycol-based solution, therefore, we tried to cryopreserve immature oocytes by vitrification. During cooling with liquid nitrogen, all intact oocytes became opaque, but many AQP3-expressing oocytes remained transparent. This indicates that the expression of AQP3 is effective in preventing intracellular ice from forming during cooling. During warming, however, all the AQP3-expressing oocytes became opaque, indicating that intracellular ice formed. Therefore, the dehydration and permeation by propylene glycol were still insufficient. Further studies are necessary to realize the cryopreservation of fish oocytes.     

Bovine pulmonary alveolar macrophages: antemortem recovery and in vitro evaluation of bacterial phagocytosis and killing

A system was developed to recover pulmonary alveolar macrophages (PAM) from living cattle and to evaluate the function of these cells by measuring bacterial phagocytosis and killing. For the collection of PAM, single-tube and telescoped double-tube pulmonary lavage devices were compared. The total recovery, using these systems, was 70 +/- 10.7% of infused fluid, yielding approximately 87% PAM. The total number of cells per collection was approximately 5 times higher with the single-tube device (6.87 +/- 0.78 x 10(7) cell/ml) than with the telescoped double-tube device (1.3 +/- 0.1 X 10(7) cells/ml). Phagocytosis and intracellular killing of Staphylococcus epidermidis and Staphylococcus aureus by PAM in media suspension and by plastic-adherent PAM were evaluated. In addition, different bacteria-to-macrophage ratios were assessed, as well as the intracellular killing of S epidermidis at periodic intervals. Results showed that over a 3-hour period, similar numbers of both bacteria were phagocytized, but intracellular killing of S epidermidis was more efficient than intracellular killing of S aureus. It also was found (i) that suspended PAM and adherent PAM phagocytized similar numbers of bacteria; (ii) that when the bacteria-to-cell ratio was 10:1, the numbers of phagocytized bacteria and intracellular killing were higher than when the ratio was 1:10; and (iii) that killing of S epidermidis by adherent PAM was directly proportional to incubation time. The time that PAM are in culture affects the phagocytosis and killing of intracellular bacteria, as shown by increased phagocytosis and by intracellular killing of S epidermidis by PAM in suspension for 48 hours or plastic adherent for 60 hours after collection.     

Phagocytosis and intracellular killing of Campylobacter jejuni by elicited chicken peritoneal macrophages.

In vitro phagocytosis and intracellular survival of Campylobacter jejuni (strains B540 and Clin 1) in chicken peritoneal macrophages were studied. Macrophages were induced with Sephadex G-50 and harvested 48 hr later by peritoneal lavage. The extent of phagocytosis over time was determined by enumerating the intracellular C. jejuni after removal of extracellular C. jejuni with gentamicin. Pre-incubation of C. jejuni with antiserum generally enabled the macrophages to ingest greater numbers of cells than when the organism was pre-incubated in phosphate-buffered saline. C. jejuni were exposed to macrophage uptake for 30 minutes in a 5% CO2 incubator at 42 C. This suspension was then exposed to 12.5 micrograms gentamicin/ml to eliminate extracellular bacteria. Subsequently, the intracellular survival of C. jejuni was examined by monitoring its number within the macrophage at 30 minutes, 3 hr, and 6 hr after phagocytosis. Macrophages from C. jejuni-colonized chickens and from uncolonized control chickens were able to almost destroy the organism within the experimental period.     

Alternative activation modifies macrophage resistance to Mycobacterium bovis

The aim of this study was to evaluate the influence of macrophage alternative activation in the intracellular pathogen natural disease resistance phenotype of the host. Macrophage monolayers from resistant (R) (3) or susceptible (S) (3) cattle donors were treated with 10 ng/ml of bovine recombinant IL-4 (rbIL-4), and infected with virulent and avirulent Mycobacterium bovis (MOI 10:1). Bactericidal assays were performed to assess the bacterial phagocytic index and intracellular survival. Total RNA was reverse transcribed and used to analyze the relative changes in gene expression of IL-10, IL-12, IL-18 IL-1β, TNF-α, MCP-1, MCP-2, IL-6, MIP-1, MIP-3, iNOS, ARGII and SLAM by real time PCR. Cell supernatants were collected and nitric oxide and arginase production was assessed. Apoptosis induction was measured by TUNEL. IL-4 treatment increased the phagocytic index in both R and S macrophages; however intracellular survival was augmented mainly in S macrophages. Alternative activation decreased gene expression of pro-inflammatory cytokines, nitric oxide production and DNA fragmentation mainly in R macrophages. On the other hand, arginase production was not different between R and S macrophages. Alternative activation modifies the macrophage response against M. bovis. IL-4 treatment minimized the functional differences that exist between R and S macrophages.     

Bta‐miR‐152 affects intracellular triglyceride content by targeting the UCP3 gene

According to our previous studies, bta‐miR‐152, PRKAA1 and UCP3 are differentially expressed in mammary gland tissues of high milk fat and low milk fat cows, and the trend in bta‐miR‐152 expression is opposite from those of PRKAA1 and UCP3. To further identify the function and regulatory mechanism of bta‐miR‐152 in milk fat metabolism, we investigated the effect of bta‐miR‐152 on cellular triglyceride content in bovine mammary epithelial cells cultured in vitro, on the basis of bta‐miR‐152 overexpression and inhibition assays. The target genes of bta‐miR‐152 were identified through qPCR, Western blotting and dual luciferase reporter gene detection. Compared with that in the control group, the expression of UCP3 was significantly lower in the bta‐miR‐152 mimic group, the expression of PRKAA1 was decreased, and the intracellular TAG content was significantly increased. After transfection with bta‐miR‐152 inhibitor, the expression of UCP3 increased significantly, and the expression of PRKAA1 decreased, but the difference was not significant; in addition, the intracellular TAG content decreased significantly. Therefore, we concluded that bta‐miR‐152 affects the intracellular TAG content by targeting UCP3.     

Pharmacokinetics of gemcitabine and its primary metabolite in dogs after intravenous bolus dosing and its in vitro pharmacodynamics

Gemcitabine is a chemotherapeutic agent used to treat a variety of cancers in humans and dogs. In this study, the plasma pharmacokinetics of gemcitabine and its inactive metabolite, 2',2'-difluorodeoxyuridine (dFdU), were investigated in dogs after intravenous bolus gemcitabine doses of 3, 10, and 30 mg/kg. Furthermore, the intracellular accumulation of the active metabolite gemcitabine triphosphate, as a surrogate pharmacodynamic endpoint, was also determined in vitro in canine melanoma cells. Gemcitabine was characterized by linear kinetics, while dFdU dose proportionality remains unknown. The average gemcitabine clearance was 0.560 L/h.kg and volume of distribution at steady-state of 1.27 L/kg. The average terminal elimination half-life, depending on dose, ranged from 1.75 to 3.23 h. Plasma concentrations of dFdU peaked at approximately 2 h post-dosing. In vitro intracellular gemcitabine triphosphate accumulation was saturated with increasing extracellular gemcitabine concentrations. These data can be used to rationally design gemcitabine dosage regimes for canine oncology patients and as a basis for future investigations on the in vivo intracellular accumulation of gemcitabine triphosphate in dogs.     

A proposed mechanism of monensin action in inhibiting ruminal bacterial growth: effects on ion flux and protonmotive force

Streptococcus bovis, a Gram-positive ruminal bacterium, was unable to grow in the presence of monensin. When monensin (5 mg/liter) was added to actively growing cultures, there was an immediate decrease in growth rate, and within 3 h no further growth was observed. Glucose utilization and lactate production continued for another 8 h even though growth had ceased. Monensin caused a decrease (P less than .05) in intracellular K+, a decrease (P less than .05) in intracellular pH and an increase (P less than .05) in intracellular Na+. The net exchange of K+ for Na+ and H+ via monensin was driven by the difference in concentration of K+ and Na+ across the cell membrane. Non-treated cells maintained a 70-fold gradient (inside higher) for K+, while the Na+ gradient was only 2.7-fold (inside higher). Previous models were based on a reverse mechanism whereby monensin would drive an efflux of Na+ from ruminal bacteria, and were not supported by experimental measurements of intracellular Na+ and K+.     

Using 3D animations to teach intracellular signal transduction mechanisms: taking the arrows out of cells

Traditional methods of teaching intracellular biological processes and pathways use figures or flowcharts with the names of molecules linked with arrows. Many veterinary students, presented with such material, simply memorize the names or chemical structures of the molecules and are then likely to forget the material once the examination is completed. To address this problem, the authors designed, created, and field-tested new teaching media that incorporate realistic three-dimensional (3D) animations depicting the dynamic changes that occur in intracellular molecules during cellular activation. Testing found that veterinary students taught using traditional teaching media (e.g., lectures, handouts, textbooks) are proficient in memorizing the names and order of intracellular molecules but unable to appreciate the interactions between these elements or their spatial relationships within cells. In contrast, more than 90% of veterinary students taught using 3D animations not only recall the facts about the intracellular elements but also develop accurate mental images of the interactions among these molecules and their spatial relationships. These findings strongly suggest that the comprehension of complex biological processes by veterinary students can be enhanced by the use of dynamic 3D depictions of these processes in the classroom.     

Assessment of intracellular Ca2+, cAMP and 1,2-diacylglycerol in cryopreserved buffalo (Bubalus bubalis) spermatozoa on supplementation of taurine and trehalose in the extender

In mammalian spermatozoa, intracellular calcium plays a major role in sperm functions like motility and capacitation. Cryopreservation-induced modifications to sperm membrane result in an influx of intracellular calcium affecting calcium-dependent intracellular signalling pathways. Intracellular calcium activates adenyl cyclase to produce cAMP that activates phospholipase A(2) (PLA(2) ) and phospholipase C (PLC) generating lysophosphatidyl choline, 1,2-diacylglycerol (DAG) and IP(3) , acting as intracellular secondary messengers required for sperm capacitation. Present study was designed to determine levels of intracellular calcium, cAMP and DAG in fresh and frozen-thawed buffalo spermatozoa cryopreserved in the presence and absence of taurine or trehalose. A total number of nine ejaculates from three randomly chosen buffalo bulls were cryopreserved in Tris-based egg yolk extender and thawed in warm water at 37°C. The cAMP was measured by enzyme immuno assay, and intracellular calcium was quantified using fluorescent dye FURA 2-AM. Total lipid was extracted from spermatozoa, and DAG was estimated using thin layer chromatography followed by spectrophotometric analysis. Intracellular calcium, cAMP and DAG levels in spermatozoa were significantly (p < 0.01) increased following cryopreservation as compared to fresh ejaculate. Addition of taurine or trehalose to the freezing medium significantly decreased (p < 0.01) the levels of intracellular calcium and cAMP in frozen-thawed spermatozoa. 1,2-diacylglycerol content was also decreased significantly (p < 0.01) in spermatozoa cryopreserved in presence of additives. Moreover, significant (p < 0.01) improvement in post-thaw motility, viability and membrane integrity of spermatozoa on addition of taurine or trehalose clearly indicated the reduced level of capacitation-like changes in buffalo spermatozoa.     

Determination of intracellular reactive oxygen species and high mitochondrial membrane potential in Percoll-treated viable boar sperm using fluorescence-activated flow cytometry

The use of frozen semen in the swine industry is limited by problems with viability and fertility compared with liquid semen. Part of the reduction in sperm motility and fertility associated with cryopreservation may be due to oxidative damage from excessive or inappropriate formation of reactive oxygen species (ROS). Chemiluminescence measurements of ROS are not possible in live cells and are problematic because of poor specificity. An alternative approach, flow cytometry, was developed to identify viable boar sperm containing ROS utilizing the dyes hydroethidine and 2', 7'-dichlorodihydrofluorescein diacetate as oxidizable substrates and impermeant DNA dyes to exclude dead sperm. The percentage of sperm with high mitochondrial transmembrane potential was determined by flow cytometry using the mitochondrial probe 5, 5', 6, 6'-tetrachloro-1, 1', 3, 3'-tetraethylbenzimidazolylcarbocyanine iodide with propidium iodide staining to exclude nonviable cells. Sperm were incubated with and without ROS generators and free radical scavengers. Basal ROS formation was low (less than 4%) and did not differ (P = 0.26) between viable fresh and frozen-thawed boar sperm. In addition, fresh and frozen-thawed viable sperm were equally susceptible (P = 0.20) to intracellular formation of ROS produced by xanthine/xanthine oxidase (94.4 and 87.9% of sperm, respectively). Menadione increased (P < 0.05) ROS formation, decreased (P < 0.05) JC-1-aggregate fluorescence intensity, and decreased (P < 0.05) motion variables by 25 to 60%. The mechanism of inhibition of motility by ROS formation may be related to a decrease in mitochondrial charge potential below a critical threshold. Catalase and superoxide dismutase treatment in the presence of xanthine/xanthine oxidase indicated that hydrogen peroxide was the primary intracellular ROS measured. Further, catalase, but not superoxide dismutase, was capable of attenuating ROS-induced inhibition of motility. Whereas basal intracellular hydrogen peroxide formation was low in viable fresh and frozen-thawed boar sperm, both were quite susceptible to external sources of hydrogen peroxide.     

Survival of Salmonella serovar Typhimurium inside porcine monocytes is associated with complement binding and suppression of the production of reactive oxygen species

The development of the carrier state in swine after infection with Salmonella serovar Typhimurium (S. Typhimurium) has not been elucidated yet. Possibly, phagocytes like macrophages play a crucial role. It was the aim of the present study to characterize the interaction of a S. Typhimurium strain and its hilA and ssrA mutants with porcine peripheral blood monocytes (PBM). Exposure of porcine PBM to S. Typhimurium induced the production of reactive oxygen species (ROS), requiring bacterial protein synthesis. The numbers of intracellular bacteria sharply decreased over a period of 3h. Monocytes obtained from different pigs differed markedly in their ROS production and in their ability to kill the bacteria. Interestingly, high ROS production did not coincide with increased intracellular killing. Using diphenylene iodonium inhibition of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activity, it was shown that bacterial killing was ROS-dependent only within 1h post inoculation, but was ROS-independent from 1h post inoculation onwards. This might be explained by the finding that metabolically active Salmonella bacteria were capable of suppressing the respiratory burst activity in a SPI-1- and SPI-2-independent manner without causing measurable cell damage. Opsonization with complement did not alter the ROS production. Nevertheless, it increased intracellular survival of the bacteria. In conclusion, survival of S. Typhimurium inside porcine PBM is promoted by suppression of respiratory burst activity and complement binding.